Absence Of Epidermal Growth Factor Research Articles

107 PRODUCTION OF EPIDERMAL GROWTH FACTOR BY BOVINE ENDOMETRIAL CELLS AND ITS EFFECTS ON EMBRYONIC INTERFERONT AND PROSTAGLANDIN

A dynamic interaction between bioactive products of the embryo (blastocyst) and the endometrium is crucial for the successful establishment of pregnancy. In ruminants, the principal signal for maternal recognition of pregnancy is interferon-τ (IFNT) secreted by the trophoectoderm between Days 8 and 20 post-fertilization. Epidermal growth factor (EGF) produced by the endometrium acting through EGF receptors (EGFR) present in the blastocyst seems to regulate embryonic production of IFNT. Epidermal growth factor and IFNT have been shown to play crucial roles in controlling luteolytic prostaglandin (PG) F2α (PGF) and luteotropic PGE2 production by bovine endometrium. However, it is unknown how these bioactive molecules regulate uterine function during maternal recognition of pregnancy. To clarify the main source of EGF in bovine endometrium and the mechanisms regulating the interaction between the hatched blastocyst and maternal uterine environment, the production of EGF by cultured endometrial epithelial and stromal cells and the effects of EGF on embryonic IFNT and PG were investigated. In addition, the effects of EGF on PGE2 and PGF production by cultured epithelial or stromal cells were examined. Endometrial epithelial and stromal cells were enzymatically isolated on the day of ovulation, seeded at a density of 100 000 viable cells mL–1, and cultured at 38°C in a humidified atmosphere of 5% CO2 in air. After the cells reached 90% confluence, they were cultured in the presence or absence of EGF (0.1, 1.0, 10, and 100 ng mL–1) for 24 h. Cells cultured in the absence of EGF and their cultured media were collected separately for protein analysis. Hatched bovine blastocysts (Days 8–10) were also cultured and exposed to EGF (1, 10, and 100 ng mL–1) for 24 h. Protein concentrations of EGF and IFNT in the cultured media were determined by commercial enzyme immunoassay kit. Hormonal concentrations were analysed by ANOVA followed by Fisher's protected least-significant difference procedure (PLSD) as a multiple comparison test by StatView (Abacus Concepts Inc., Berkeley, CA, USA). The concentration of EGF in the culture media of epithelial cells cultured in the absence of EGF was significantly (P < 0.05) higher than in the cultured media of endometrial stromal cells. Epidermal growth factor (10 and 100 ng mL–1) increased embryonic production of IFNT and luteotropic PGE2 production but not luteolytic PGF by hatched blastocyst. EGF (100 ng mL–1) increased both PGE2 and PGF production (P < 0.05) by cultured endometrial epithelial and stromal cells. The overall results suggest that endometrial epithelial cells rather than stromal cells are the main source of EGF. Epidermal growth factor produced by epithelial cells stimulates the production of IFNT by bovine trophoblasts. The capacity of conceptus to increase IFNT and luteotropic PGE2 production rather than luteolytic PGF in response to EGF stimulation may be essential for the establishment of pregnancy in cattle.

Recruitment of the Adaptor Protein Grb2 to EGFR Tetramers

Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM–FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2–mRFP) and EGFR (EGFR–eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2–mRFP with EGFR–eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR–eGFP per cluster. In contrast, the pool of EGFR–eGFP without Grb2–mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR–eGFP and Grb2–mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit.

Effects of epidermal growth factor on the invasive activity and cytoskeleton of oral squamous cell carcinoma cell lines.

Epidermal growth factor (EGF) is present at high concentrations in human saliva and modulates the growth and differentiation of various cancer cells. To elucidate the molecular mechanisms by which EGF affects oral cancer proliferation and invasion, the current study analyzed the Matrigel invasion activity of cultured oral cancer cell lines. Cell proliferation under the influence of EGF was subjected to Matrigel invasion assays, and cell proliferation in the absence of EGF was used as control. Northern blot analyses quantified the invasiveness and tumorigenicity. Chloramphenicol acetyltransferase assay determined the EGF stimulation of matrix metalloproteinase (MMP) 1 expression. EGF increased the number of cells penetrating the Matrigel membrane. Northern blot analysis revealed that MMP1 and cytokeratin 19 expression correlate with EGF. In addition, the morphology of HSC-3 and SAS cells changed following the addition of EGF to the culture medium. A transient transfection assay revealed that EGF increases the promoter activities of MMP1 in HSC-3 cells. These observations suggested that EGF increases the invasive activity of oral cancer cells, partly by increasing MMP1, and morphological changes may be induced by altering the composition of cytoskeletal proteins.

Abstract B280: Histone deacetylase (HDAC) 6 inhibition potentiates the cytotoxicity of lapatinib and PI3K inhibitors on Her2+ breast cancer cell lines.

Abstract ACY-1215 is a novel and selective oral inhibitor of HDAC6 currently in Phase I clinical trials in combination with bortezomib or lenalidomide in relapsed/refractory multiple myeloma. Inhibitors of HDAC6 have demonstrated anti-cancer proliferative activity in various cancer types, including Her2+ breast cancer. Blocking HDAC6 activity has been shown to cause Her2+ breast cancer cell growth inhibition through various mechanisms, including destabilization of Her2 mRNA and protein. In the present work, the impact of HDAC6 inhibition has been investigated on Her2+ breast cancer cell growth and cell migration using HDAC6 inhibitor alone or in combination with either a Her2 inhibitor (lapatinib) or a PI3K inhibitor (GDC-0941, IPI-145 and CAL-101). Anti-tumor activity of ACY-1215 was investigated using a panel of nearly 100 human tumor cell lines derived from the major cancer types and Her2+ breast cancer cells were among the most sensitive cells to ACY-1215. Combinations of ACY-1215 with either the Her2 inhibitor lapatinib or PI3K inhibitor GDC-0941 demonstrated synergistic cytotoxicity against these cancer cells. To illustrate the effects are due to HDAC6 inhibition, we used a highly HDAC6 selective inhibitor ACY-775, which has greater than 1,500 fold of selectivity against HDAC6 over Class I HDACs (HDAC1, 2 and 3), in our experiments. ACY-775 is able to sensitize Her2+ breast cancer cells MDA-MB-453 and BT-474 to lapatinib, and PI3K inhibitors (IPI-145, GDC-0941 and CAL-101). ACY-775 also blocks cancer cell migration in the presence of absence of epidermal growth factor (EGF). In agreement with the proposed mechanism of HDAC6 mediated Her2+ breast cancer cell death, ACY-1215 treatment correlated with decrease of Her2 protein, p-EGFR and p-AKT in a Her2+ breast cancer cell line MDA-MB-453. By taking advantage of our highly selective HDAC6 inhibitor ACY-775, we have shown growth and migration suppressive activities by HDAC6 inhibition in Her2+ breast cancer cells. These findings suggest selection of metastatic Her2+ breast cancer for further in vitro and in vivo validation and mechanism of action studies with ACY-1215 in combination with novel and approved standard of care therapeutics for breast cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B280. Citation Format: Min Yang, David Tamang, Simon Jones. Histone deacetylase (HDAC) 6 inhibition potentiates the cytotoxicity of lapatinib and PI3K inhibitors on Her2+ breast cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B280.

Growth hormone (GH) is a survival rather than a proliferative factor for embryonic striatal neural precursor cells

ObjectiveA possible role of GH during central nervous system (CNS) development has been suggested by the presence of this hormone and its receptor in brain areas before its production by the pituitary gland. Although several effects have been reported for GH, the specific role of this hormone during CNS development remains unclear. Here, we examined the effect of GH on proliferation, survival and neurosphere formation in primary cultures of striatal tissue from14-day-old (E14) mouse embryos. DesignGH receptor gene expression was confirmed by RT-PCR. Primary cultures of embryonic striatal cells were treated with different doses of GH in serum free media, then the number of neurospheres was determined. To examine the GH effect on proliferation and survival of the striatal primary cultures, bromodeoxyuridine (BrdU) and TUNEL immunoreactivity was conducted. ResultsIn the presence of the epidermal growth factor (EGF), GH increased the formation of neurospheres, with a maximal response at 10ng/ml, higher doses were inhibitory. In absence of EGF, GH failed to stimulate neurosphere formation. Proliferation rate in the primary striatal cultures was inhibited by 24 or 48h incubation with GH. However, in the absence of EGF, GH increased BrdU incorporation. GH treatment decreases the rate of apoptosis of nestin and GFAP positive cells in the primary striatal cultures, enhancing neurosphere formation. ConclusionsOur in vitro data demonstrate that GH plays a survival role on the original population of embryonic striatal cells, improving Neural Precursor Cells (NPCs) expansion. We suggest that this GH action could be predominant during striatal neurodevelopment.

Inhibition of cell migration by ouabain in the A549 human lung cancer cell line

The Na+/K+-ATPase α subunit is highly expressed in malignant cells. Ouabain, a cardioactive glycoside, binds to the Na+/K+-ATPase α subunit and inhibits the activity of Na+/K+-ATPase. In the present study, the effect of ouabain on the migration of A549 cells was analyzed using the wound healing and transwell chamber migration assays. The impact of ouabain on the expression of E-cadherin, N-cadherin, vimentin, matrix metalloprotease (MMP)-2 and MMP-9 was also evaluated. Ouabain treatment not only inhibited the epidermal growth factor (EGF)-enhanced migration of A549 cells, but also inhibited the basal migration of A549 cells in the absence of EGF. Ouabain decreased the overexpression of N-cadherin and vimentin induced by EGF, and decreased the expression of MMP-2 and -9 in the presence or absence of EGF. Na+/K+-ATPase is a potent therapeutic target in lung cancer and these observations indicated that the Na+/K+-ATPase inhibitor, ouabain, retards the invasion of lung cancer cells.

Abstract 3998: The IGF1 receptor/insulin receptor dual kinase inhibitor BMS-754807 targets the cancer stem cell population in addition to its synergism with Lapatinib in colorectal cancer.

Abstract Colon cancer is the second leading cause of cancer mortalities each year. Current treatment options include drugs that target the epidermal growth factor (EGF) pathway. However, the use of these drugs is limited to individuals that are wild type for KRAS. New therapies are needed for patients who develop resistance to EGF-based therapies, as well as for the 40% of patients whose tumors have mutated KRAS. Our approach has been to target pathways that regulate cancer cell metabolism. The insulin and insulin-like growth factor-1 (IGF1) pathways regulate glucose metabolism, cell growth, cell motility and anti-apoptotic pathways. In colon cancer, IGF1 and insulin enhance tumor growth, promote metastasis and increase the cancer stem cell (CSC) population. In order to target the insulin and IGF-1 pathways simultaneously, we utilized the small molecule, dual kinase inhibitor BMS-754807 which inhibits the Insulin receptor (IR) and IGF1 receptor (IGF1R) with equal potency. We previously reported that the drug effectively reduced cell viability in a panel of eight colon cancer cell lines. In combination when combined with Lapatinib, an EGFR/HER2 small molecule dual kinase inhibitor, the drug showed synergistic effects on viability in a subset of cell lines. In order to determine the mechanism of the observed synergy, we examined whether EGFR and IGF1R were interacting directly as has been observed in breast cancer. Co-immunoprecipitation experiments in colorectal cancer cells, did not demonstrate an interaction between the EGFR and IGF1R. Examination of the signaling pathways in our panel of cell lines has determined that the regulation of ERK appears to be a critical determinant in sensitivity to the combination therapy. In both of the sensitive lines, AKT was strongly activated by IGF1, while ERK was strongly activated by EGF, but less substantially by IGF1. However in the cell lines that were insensitive to the BMS-754807/Lapatinib combination, substantial activation of ERK was observed in the absence of either EGF or IGF1. Therefore constitutive ERK activation in the absence of EGF signaling may be the determinant for sensitivity to the combined therapy. Finally, we examined the efficacy of BMS-754807 in the targeting colon cancer stem cells. In our panel of eight cell lines, BMS-754807 strongly inhibited colonsphere formation from the CSC population in three cell lines, and showed moderate inhibition in four of the five remaining lines. Of interest is the fact that the cells that were most sensitive to BMS-754807 in sphere formation were the most resistant in viability assays, and did not synergize with Lapatinib. These results are encouraging as we continue to develop strategies that specifically target the cancer stem cell, and indicate that BMS-754807 has multiple potential mechanisms of action in colon cancer. Citation Format: Kristi L. Peters, David T. Dicker, Adrian Woolfson, Wafik S. El-Deiry. The IGF1 receptor/insulin receptor dual kinase inhibitor BMS-754807 targets the cancer stem cell population in addition to its synergism with Lapatinib in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3998. doi:10.1158/1538-7445.AM2013-3998

Effect of Growth Factors on the Proliferation and Gene Expression of Human Meibomian Gland Epithelial Cells

We hypothesize that growth factors, including epidermal growth factor (EGF) and bovine pituitary extract (BPE), induce proliferation, but not differentiation (e.g., lipid accumulation), of human meibomian gland epithelial cells. We also hypothesize that these actions involve a significant upregulation of genes linked to cell cycle processes, and a significant downregulation of genes associated with differentiation. Our objective was to test these hypotheses. Immortalized human meibomian gland and conjunctival epithelial cells were cultured for varying time periods in the presence or absence of EGF, BPE, EGF + BPE, or serum, followed by cell counting, neutral lipid staining, or RNA isolation for molecular biological procedures. Our studies show that growth factors stimulate a significant, time-dependent proliferation of human meibomian gland epithelial cells. These effects are associated with a significant upregulation of genes linked to cell cycle, DNA replication, ribosomes, and translation, and a significant decrease in those related to cell differentiation, tissue development, lipid metabolic processes, and peroxisome proliferator-activated receptor signaling. Serum-induced differentiation, but not growth factor-related proliferation, elicits a pronounced lipid accumulation in human meibomian gland epithelial cells. This lipogenic response is unique, and is not duplicated by human conjunctival epithelial cells. Our results demonstrate that EGF and BPE stimulate human meibomian gland epithelial cells to proliferate. Further, our findings show that action is associated with an upregulation of cell cycle and translation ontologies, and a downregulation of genetic pathways linked to differentiation and lipid biosynthesis.

Abstract 38: The role of autocrine epidermal growth factor receptor (EGFR) signaling in breast cancer metastasis

Abstract The majority of breast cancer deaths are due to the development of metastatic disease. The metastatic cascade consists of a series of steps and includes primary tumor growth and angiogenesis, invasion, intravasation, cell survival in circulation, extravasation and sustained growth at secondary organ sites to form micrometastases. Previous studies have shown that overexpression of the epidermal growth factor receptor (EGFR; ErbB1; Her1) increases tumor cell motility, intravasation and motility. In addition, it has been demonstrated that the metastatic potential of cancer cells depends not only on their intrinsic characteristics but also on their cooperative interaction with the tumor microenvironment. For instance, imposed gradients of EGF or colony-stimulating factor-1 (CSF-1) can stimulate invasion via an EGF/CSF-1 paracrine loop between tumor cells and macrophages. Because breast malignancies exhibiting increased expression of the epidermal growth factor (EGF) family of receptors and their cognate ligands are associated with more biologically aggressive disease and poorer patient prognosis, we decided to study how expression of certain members of the EGF family of ligands in the context of ErbB1 overexpression can affect the processes of invasion, intravasation and metastasis using in vitro and in vivo systems. Based on initial Oncomine cancer gene expression analysis, heparin-binding epidermal growth factor-like growth factor (HB-EGF) overexpression was correlated with more rapid recurrence of disease in breast cancer and thus, we decided to focus our study this ligand. Briefly, MTLn3 rat mammary adenocarcinoma cells and human MDA-MB 231 breast adenocarcimona cells were transduced to overexpress HB-EGF. HB-EGF expression did not significantly affect in vitro or in vivo growth rate. In vitro microchemotaxis and invasion assays to study chemotaxis and invasion, respectively, in the presence and absence of EGF, demonstrated that random motility and spontaneous invasion were both enhanced in the presence of HB-EGF expression. Interestingly, EGF-induced chemotaxis and EGF-induced in vivo invasion were both suppressed. In vivo studies show that tumor cells expressing HB-EGF not only display increased metastasis to the lungs and extravasation from circulation, but also significantly enhanced macrophage recruitment and exerted an angiogenic effect. These findings suggest that a better understanding of the role of HB-EGF in breast cancer will aid in directing and modifying therapeutic approaches aimed at inactivating the ErbB ligand/EGFR system as well as developing approaches based on the EGF family of ligands as a target for the clinical treatment of breast cancer, ultimately leading to improved patient survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 38. doi:1538-7445.AM2012-38

Abstract 3076: p53 homologue p63 trans-activates epidermal growth factor (EGF) receptor and 14-3-3α and thus promotes tumorigenesis and chemoresistance in pancreatic cancer

Abstract Pancreatic cancer is the 4th leading cause of cancer-related mortality in the United States. Despite these statistics, progress in therapy of pancreatic cancer has been slow. p53 tumor suppressor pathway is altered in 50-75% of pancreatic cancer tumors. However, the role of p53 homologs p63 and p73 has not been well studied. We previously reported that p63 is expressed in pancreatic cancer cell lines and contributes to their motility and invasion. Here we tested expression of p63 in resected tumors and investigated its role as a transcription factor in pancreatic cancer. Expression of p63 was determined by immunohistochemistry in 20 resected tumor samples (15 - adenocarcinoma, 5 - squamous) using pan-p63 (clone 4A4) and ΔNp63-specific antibodies; and by immunoblotting, real-time quantitative and semi-quantitative PCR in 5 pancreatic cancer cell lines. p63 levels were manipulated employing transient overexpression and lentiviral approaches. We studied cell doubling times, motility in a wound assay and invasiveness in Matrigel chambers in the presence or absence of epidermal growth factor (EGF). Functional reporter and chromatin immunoprecipitation assays were performed to study the effects of p63 on selected promoters. We found that the p53 homolog p63 was expressed in resected pancreatic cancer samples. Pancreatic tumors with squamous differentiation showed strong diffuse expression of p63, whereas adenocarcinomas without squamous component had a focal expression of p63 (ΔNp63). In pancreatic cancer cell lines, p63 protein levels were high in 3/5 tested cell lines and ΔNp63α was the predominantly expressed variant. By contrast, TAp63 was not detected while p73 was barely detectable in 2/5 cell lines. Ectopic expression of ΔNp63α in PANC-1 cells (which had low endogenous p63) and shRNA-mediated knockdown in T3M4 cells (with high endogenous p63) indicated that ΔNp63α promoted anchorage-dependent and independent growth, motility and invasion, and enhanced resistance to cisplatin-induced apoptosis. Motogenic effects of ΔNp63α were augmented in presence of EGF. ΔNp63α augmented the EGF receptor expression in pancreatic cancer cells via transcriptional regulation and potentiated EGF-mediated activation of ERK, Akt and JNK signaling. 14-3-3α transcription was also positively regulated by ΔNp63α and we have previously shown that 14-3-3α contributes to chemoresistance in pancreatic cancer. Thus, p53 homolog ΔNp63α is expressed in pancreatic cancer, where it enhances tumor progression and chemoresistance via trans-activation of EGF receptor and 14-3-3α. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3076. doi:1538-7445.AM2012-3076

EGF enhances the migration of cancer cells by up-regulation of TRPM7

Ion channels involved in the migration of tumor cells that is required for their invasion and metastasis. In this paper, we describe the interaction of TRPM7 channel and epidermal growth factor (EGF), an important player in cancer development in the migration of lung cancer cells. The TRPM7 currents in A549 cells were first characterized by means of electrophysiology, pharmacology and RNA interference. Removing Ca 2+ from the extracellular solution not only potentiated a large inward current, but also abolished the outward rectification. 200 μM 2-APB inhibited the outward and the inward TRPM7 currents and at the same time restored the property of outward rectification. EGF greatly enhanced the migration of A549 cells, and also markedly up-regulated the membrane protein expression of TRPM7 and the amplitude of TRPM7 currents. Depressing the function of TRPM7 with RNA interference or pharmacological agents not only reversed the EGF-enhanced migration of A549 cells but also inhibited the basal migration of A549 cells in the absence of EGF. Thus it seems that TRPM7 plays a pivotal role in the migration of A549 cells induced by EGF and thus could be a potential therapeutic target in lung cancers.

Differences between the neurogenic and proliferative abilities of Müller glia with stem cell characteristics and the ciliary epithelium from the adult human eye

Much controversy has arisen on the nature and sources of stem cells in the adult human retina. Whilst ciliary epithelium has been thought to constitute a source of neural stem cells, a population of Müller glia in the neural retina has also been shown to exhibit neurogenic characteristics. This study aimed to compare the neurogenic and proliferative abilities between these two major cell populations. It also examined whether differences exist between the pigmented and non-pigmented ciliary epithelium (CE) from the adult human eye. On this basis, Müller glia with stem cell characteristics and pigmented and non-pigmented CE were isolated from human neural retina and ciliary epithelium respectively. Expression of glial, epithelial and neural progenitor markers was examined in these cells following culture under adherent and non-adherent conditions and treatments to induce neural differentiation. Unlike pigmented CE which did not proliferate, non-pigmented CE cells exhibited limited proliferation in vitro, unless epidermal growth factor (EGF) was present in the culture medium to prolong their survival. In contrast, Müller glial stem cells (MSC) cultured as adherent monolayers reached confluence within a few weeks and continued to proliferative indefinitely in the absence of EGF. Both MSC and non-pigmented CE expressed markers of neural progenitors, including SOX2, PAX6, CHX10 and NOTCH. Nestin, a neural stem cell marker, was only expressed by MSC. Non-pigmented CE displayed epithelial morphology, limited photoreceptor gene expression and stained strongly for pigmented epithelial markers upon culture with neural differentiation factors. In contrast, MSC adopted neural morphology and expressed markers of retinal ganglion cells and photoreceptors when cultured under similar conditions.This study provides the first demonstration that pigmented CE possess different proliferative abilities from non-pigmented CE. It also showed that although non-pigmented CE express genes of retinal progenitors, they do not differentiate into neurons in vitro, as that seen with Müller glia that proliferate indefinitely in vitro and that acquire markers of retinal neurons in culture under neural differentiation protocols. From these observations it is possible to suggest that Müller glia that express markers of neural progenitors and become spontaneously immortalized in vitro constitute a potential source of retinal neurons for transplantation studies and fulfil the characteristics of true stem cells due to their proliferative and neurogenic ability.

Role of β4 integrin phosphorylation in human invasive squamous cell carcinoma: regulation of hemidesmosome stability modulates cell migration

Hemidesmosomes (HDs) are multiprotein structures that anchor epithelia to the basement membrane. During squamous cell carcinoma (SCC) invasion, there is a reduction in the number of HDs, which may facilitate dissemination. Mechanisms of HD disassembly are incompletely understood. Previous work has shown that epidermal growth factor (EGF)-induced phosphorylation of the β4 integrin on three of its serines, S1356S1360S1364, can induce HD disassembly in normal cells. Here, we examine the role of β4 integrin serine phosphorylation in SCC. We have found that around 60% of invasive cutaneous SCC show increased β4 phosphorylation on S1356 when compared with carcinoma in situ or normal tissue. To assess the mechanisms by which SCC increases β4 phosphorylation, we performed in vitro analyses. Compared with keratinocytes, SCC cells showed increased levels of S1356 phosphorylation in the absence of EGF, correlating with reduced HD-like structures. In addition, phospho-S1356 signal was largely segregated from other HD components. Epidermal growth factor receptor and PKC inhibitors inhibited basal levels of S1356 phosphorylation in SCC, suggesting that cells use intrinsic mechanisms to activate the EGF signaling pathway to induce β4 phosphorylation. Moreover, these inhibitors stabilized HD-like structures in SCC cells and reduced their migratory ability. Mutation of S1356S1360S1364 in SCC cells to non-phosphorylatable alanines stabilized HD-like structures and substantially reduced migration, while mutation into phosphorylation mimicking aspartate reduced HD-like structures but had no effect on migration, suggesting that serine phosphorylation function is releasing anchorage rather than promoting migration. Altogether these results suggest that β4 serine phosphorylation may have an important role during SCC invasion by destabilizing HDs and facilitating migration.

Epidermal growth factor induces adult human islet cell dedifferentiation

Given the inherent therapeutic potential of the morphogenetic plasticity of adult human islets, the identification of factors controlling their cellular differentiation is of interest. The epidermal growth factor (EGF) family has been identified previously in the context of pancreatic organogenesis. We examined the role of EGF in an in vitro model whereby adult human islets are embedded in a collagen gel and dedifferentiated into duct-like epithelial structures (DLS). We demonstrated that DLS formation was EGF dependent, while residual DLS formation in the absence of added EGF was abrogated by EGF receptor inhibitor treatment. With respect to signaling, EGF administration led to an increase in c-Jun NH2-terminal kinase (JNK) phosphorylation early in DLS formation and in AKT and extracellular signal-regulated kinase (ERK) phosphorylation late in the process of DLS formation, concomitant with the increased proliferation of dedifferentiated cells. In the absence of EGF, these phosphorylation changes are not seen and the typical increase in DLS epithelial cell proliferation seen after 10 days in culture is attenuated. Thus, in our model, EGF is necessary for islet cell dedifferentiation, playing an important role in both the onset of DLS formation (through JNK) and in the proliferation of these dedifferentiated cells (through AKT and ERK).

MUC5AC Expression through Bidirectional Communication of Notch and Epidermal Growth Factor Receptor Pathways

Hyperproduction of goblet cells and mucin in the airway epithelium is an important feature of airway inflammatory diseases. We investigated the involvement of Notch signaling in MUC5AC expression in NCI-H292 cells, a human lung carcinoma cell line. Epidermal growth factor (EGF) stimulated generation of the Notch intracellular domain (NICD) in a RBP-Jκ-dependent manner. Treatment with γ-secretase inhibitors L-685,458 or DAPT or introduction of small interfering RNA directed against Notch1 reduced EGF-induced MUC5AC expression. The inhibitory effect of L-685,458 on EGF-induced MUC5AC mRNA and protein expression was also observed in primary human bronchial epithelial cells. Blockage of Notch signaling with L-685,458 or Notch siRNA resulted in a decrease in EGF-induced phosphorylation of ERK. These results suggested that ERK activation is necessary for the regulation of EGF receptor (EGFR)-mediated MUC5AC expression by Notch signaling. Conversely, forced expression of NICD induced both EGFR and ERK phosphorylation with MUC5AC expression even in the absence of EGF. Treatment of the NICD-expressing cells with EGF further augmented ERK phosphorylation in an additive manner. The ERK phosphorylation induced by exogenous NICD was inhibited by treatment with an Ab that antagonizes EGFR activity as well as by inhibitors of EGFR and ERK, implying that Notch signaling induces MUC5AC expression by activating the EGFR pathway. Collectively, these results suggest that MUC5AC expression is regulated by a bidirectional circuit between Notch and EGFR signaling pathways.

Growth hormone and prolactin regulate human neural stem cell regenerative activity

We have previously shown that the growth hormone (GH)/prolactin (PRL) axis has a significant role in regulating neuroprotective and/or neurorestorative mechanisms in the brain and that these effects are mediated, at least partly, via actions on neural stem cells (NSCs). Here, using NSCs with properties of neurogenic radial glia derived from fetal human forebrains, we show that exogenously applied GH and PRL promote the proliferation of NSCs in the absence of epidermal growth factor or basic fibroblast growth factor. When applied to differentiating NSCs, they both induce neuronal progenitor proliferation, but only PRL has proliferative effects on glial progenitors. Both GH and PRL also promote NSC migration, particularly at higher concentrations. Since human GH activates both GH and PRL receptors, we hypothesized that at least some of these effects may be mediated via the latter. Migration studies using receptor-specific antagonists confirmed that GH signals via the PRL receptor promote migration. Mechanisms of receptor signaling in NSC proliferation, however, remain to be elucidated. In summary, GH and PRL have complex stimulatory and modulatory effects on NSC activity and as such may have a role in injury-related recovery processes in the brain.

Activation of Extracellular-Signal Regulated Kinase by Epidermal Growth Factor Is Potentiated by cAMP-Elevating Agents in Primary Cultures of Adult Rat Hepatocytes

We investigated the effects of α- and β-adrenergic agonists on epidermal growth factor (EGF)-stimulated extracellular-signal regulated kinase (ERK) isoforms in primary cultures of adult rat hepatocytes. Hepatocytes were isolated and cultured with EGF (20 ng/ml) and/or α(1)-, α(2)- and β(2)-adrenergic agonists. Phosphorylated ERK isoforms (ERK1; p44 mitogen-activated protein kinase (MAPK) and ERK2; p42 MAPK) were detected by Western blotting analysis using anti-phospho-ERK1/2 antibody. The results show that EGF induced a 2.5-fold increase in ERK2-, but not ERK1-, phosphorylation within 3 min. This EGF-induced ERK2 activation was abolished by treatment with the EGF-receptor kinase inhibitor AG1478 (10(-7) M) or the MEK (MAPK kinase) inhibitor PD98059 (10(-6) M). The α(2)-adrenergic and β(2)-adrenergic agonists, UK14304 (10(-6) M) and metaproterenol (10(-6) M), respectively, had no effect in the absence of EGF, but metaproterenol significantly potentiated EGF-induced ERK2 phosphorylation. Moreover, the cell-permeable cAMP analog 8-bromo cAMP (10(-7) M), also potentiated EGF-induced ERK2 phosphorylation. The effects of these analogs were antagonized by the protein kinase A (PKA) inhibitor H-89 (10(-7) M). These results suggest that direct or indirect activation of PKA represents a positive regulatory mechanism for EGF stimulation of ERK2 induction.

P13. Wnt signaling pathways regulate epithelial polarity and morphogenesis

Wnt proteins are highly conserved secreted proteins that are essential for development in all metazoan organisms. Wnt proteins bind to cell surface receptors and activates the β-catenin-dependent and -independent pathways. The β-catenin-dependent pathway regulates cell proliferation and differentiation through the regulation of target gene expression. The β-catenin-independent pathway is involved in cell migration and polarity. Knockout mouse studies revealed that both the β-catenin-dependent and -independent pathways of Wnt signaling are important in the epithelial morphogenesis of tubular organs such as the lungs, kidneys, guts, and mammary glands, but its molecular mechanism is poorly understood. Epithelial cells established apical–basal polarity and formed cyst with central lumen in three-dimensional culture. Epidermal growth factor (EGF) increased the size of cyst through the promotion of cell proliferation. Although Wnt3a, which is a representative ligand that activates the β-catenin-dependent pathway, did not affect cyst morphogenesis, combination of Wnt3a and EGF induced tube-like branching morphogenesis. Treatment with EGF and blebbistatin, a Myosin II inhibitor, induced branching morphogenesis similar to that induced by EGF and Wnt3a. Blebbistatin inhibited phosphorylation of paxillin, which is a component of focal adhesions. Knockdown of β-catenin or LRP6, which is a receptor of Wnt3a, inhibited branching morphogenesis induced by Wnt3a. Wnt5a, which is a representative ligand that activates the β-catenin-independent pathway, did not affect cyst morphology in the presence or absence of EGF. Knockdown of Wnt5a inhibited the formation of apical membrane domain of epithelial cells. ECM signaling from the basal cell surface is important for the decision of apical–basal polarity. Wnt5a was observed at the basal cell surface and Ror2, a Wnt5a receptor specifically localized basolateral cell surface in epithelial cyst. These results indicate that both the β-catenin-dependent and -independent pathways of Wnt signaling are involved in the regulation of epithelial polarity and morphogenesis through the distinct mechanisms.

Intracellular calcium as a second messenger following growth stimulation of human keratinocytes.

The mitogenic effect of the neuropeptide substance P and bombesin was investigated in normal human keratinocytes in serum-free culture, both with and without the presence of epidermal growth factor (EGF). Although both neuropeptides induced a small increase in cell numbers in the presence of EGF, the response was much greater in its absence, and cell numbers increased to 200% of controls at 5 days. Changes in intracellular free calcium are frequently seen following mitogenic stimulation of cells, and this phenomenon was studied in individual keratinocytes. Epidermal growth factor (10 ng/ml) induced calcium transients in 57% (n = 21) of cells. The mean intracellular free calcium was 97 +/- 11 nM (mean +/- SEM) in quiescent cells, and the calcium transients reached approximately 250 nM for 3-4 min. In the presence of EGF, calcium transients were never observed with the addition of either substance P or bombesin. For EGF-deprived cultures, 20% of keratinocytes (n = 10) showed a large calcium transient following the addition of 500 nM bombesin, and 63% (n = 12) of cells gave calcium transients following the addition of 700 nM of substance P. Studies in calcium-free medium, and following depletion of intracellular calcium stores with thapsigargin, showed that all of the calcium transients were dependent on the presence of intracellular stores, but also partially mediated by an influx of extracellular calcium. These studies demonstrate the mitogenic effect of substance P and bombesin on human keratinocytes in the absence of EGF. The ability of the neuropeptides to increase keratinocyte growth in culture suggests a possible in wound healing.

Asp-960/Glu-961 Controls the Movement of the C-terminal Tail of the Epidermal Growth Factor Receptor to Regulate Asymmetric Dimer Formation

The epidermal growth factor (EGF) receptor is a tyrosine kinase that dimerizes in response to ligand binding. Ligand-induced dimerization of the extracellular domain of the receptor promotes formation of an asymmetric dimer of the intracellular kinase domains, leading to stimulation of the tyrosine kinase activity of the receptor. We recently monitored ligand-promoted conformational changes within the EGF receptor in real time using luciferase fragment complementation imaging and showed that there was significant movement of the C-terminal tail of the EGF receptor following EGF binding (Yang, K. S., Ilagan, M. X. G., Piwnica-Worms, D., and Pike, L. J. (2009) J. Biol. Chem. 284, 7474-7482). To investigate the structural basis for this conformational change, we analyzed the effect of several mutations on the kinase activity and luciferase fragment complementation activity of the EGF receptor. Mutation of Asp-960 and Glu-961, two residues at the beginning of the C-terminal tail, to alanine resulted in a marked enhancement of EGF-stimulated kinase activity as well as enhanced downstream signaling. The side chain of Asp-960 interacts with that of Ser-787. Mutation of Ser-787 to Phe, which precludes this interaction, also leads to enhanced receptor kinase activity. Our data are consistent with the hypothesis that Asp-960/Glu-961 represents a hinge or fulcrum for the movement of the C-terminal tail of the EGF receptor. Mutation of these residues destabilizes this hinge, facilitating the formation of the activating asymmetric dimer and leading to enhanced receptor signaling.